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The Role of Hexokinase and Hexose Transporters in Preferential Use of Glucose Over Fructose and Downstream Metabolic Pathways in the Yeast Yarrowia Lipolytica

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The Role of Hexokinase and Hexose Transporters in Preferential Use of Glucose Over Fructose and Downstream Metabolic Pathways in the Yeast Yarrowia Lipolytica International Journal of Molecular Sciences Article The Role of Hexokinase and Hexose Transporters in Preferential Use of Glucose over Fructose and Downstream Metabolic Pathways in the Yeast Yarrowia lipolytica Piotr Hapeta 1 , Patrycja Szczepa ´nska 1, Tadeusz Witkowski 1, Jean-Marc Nicaud 2 , Anne-Marie Crutz-Le Coq 2 and Zbigniew Lazar 1,* 1 Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Chełmo´nskiegoStreet 37, 51-630 Wrocław, Poland; [email protected] (P.H.); [email protected] (P.S.); [email protected] (T.W.) 2 Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78352 Jouy-en-Josas, France; [email protected] (J.-M.N.); [email protected] (A.-M.C.-L.C.) * Correspondence: [email protected] Abstract: The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We Citation: Hapeta, P.; Szczepa´nska,P.; investigated the role of hexose transporters and hexokinase (native and fructophilic) in this prefer- Witkowski, T.; Nicaud, J.-M.; ence. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the Crutz-Le Coq, A.-M.; Lazar, Z. The glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Role of Hexokinase and Hexose Interestingly, combined overexpression of either hexokinase with hexose transporters significantly Transporters in Preferential Use of accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion Glucose over Fructose and of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in Downstream Metabolic Pathways in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with the Yeast Yarrowia lipolytica. Int. J. Mol. hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but Sci. 2021, 22, 9282. https://doi.org/ 10.3390/ijms22179282 also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L). Academic Editor: Ismail Fliss Keywords: Yarrowia lipolytica; hexokinase; hexose transporters; glucose; fructose; erythritol Received: 25 July 2021 Accepted: 25 August 2021 Published: 27 August 2021 1. Introduction The oleaginous yeast Yarrowia lipolytica is an industrial micro-organism used for Publisher’s Note: MDPI stays neutral production of complex biological compounds such as tailor-made lipids [1,2], biofuel pre- with regard to jurisdictional claims in cursors [3] or industrial and therapeutic enzymes [4,5], as well as more compact molecules published maps and institutional affil- such as acidifying agent citric acid [6] and low-calorie sweeteners, including erythritol and iations. mannitol [7,8]. Substrates can be chosen among hydrophobic carbon sources (n-alkanes, oils and fats) and hydrophilic ones, such as glycerol, monosaccharides and organic acids. From the latter, glycerol is a superior carbon source for Y. lipolytica, due to its rapid and preferred use over glucose when supplied in a mixture [9–11]. To date, production of Copyright: © 2021 by the authors. erythritol by this yeast relies on the use of glycerol in the culture medium as it acts as a Licensee MDPI, Basel, Switzerland. stress factor, increasing osmotic pressure required for erythritol overproduction [12]. In This article is an open access article turn, the narrow spectrum of monosaccharides used by wild-type Y. lipolytica strains, which distributed under the terms and includes glucose, fructose and mannose [13], can be extended by genetic engineering to conditions of the Creative Commons galactose and xylose [14–16]. Glucose and fructose are readily available substrates present Attribution (CC BY) license (https:// in plant biomass and industrial by-products such as molasses [17]. Many yeast species, creativecommons.org/licenses/by/ including Saccharomyces cerevisiae, show a preferential uptake of glucose over fructose in 4.0/). Int. J. Mol. Sci. 2021, 22, 9282. https://doi.org/10.3390/ijms22179282 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9282 2 of 20 mixed sugar media [18]. This trait is particularly prominent in Y. lipolytica, for which sev- eral experiments enlightened that fructose uptake begins only after glucose is completely or mostly depleted from the medium [19,20]; the molecular mechanism behind remains unknown. In order to enter the metabolism, monosaccharides need first to be taken up by the cell, a step which requires transporters, then to be activated through phosphorylation by hexose kinases. Y. lipolytica possesses six bona fide hexose transporters (encoded by the genes YHT1 to YHT6) belonging to the 24-protein Sugar Porter (SP) family [20]. Among them, Yht1, Yht3 and Yht4 are efficient broad-range hexose transporters enabling glucose, fructose, mannose and galactose uptake as determined by the heterologous complementation study [20]. Yht1 and Yht4 were identified as the main active hexose transporters in this yeast as deletion of both genes prevented growth on the four hexoses [20]. Furthermore, out of the six YHT genes, only YHT1 (YALI0C06424g) and YHT4 (YALI0E23287g) were consistently transcribed during the growth of W29 and H222 strains in glucose- and fructose-based media [20]. These were also the only Yht transporters expressed in media containing glucose, glycerol and a mixture of both under nitrogen-limiting conditions, showing a differential expression pattern depending on carbon source and nitrogen concentration [9]. YHT3 (YALI0F19184g) appeared to be poorly transcribed in the conditions tested. Moreover, the strain-dependent polymorphism in this gene had an impact on the efficiency of the transporter as the gene in Y. lipolytica H222 encoded a much more efficient transporter than its counterpart from the W29 strain [20]. Two enzymes with hexokinase activity have been identified in Y. lipolytica. One of them is a glucokinase, with glucose as the only substrate; the other one phosphorylates glucose, fructose and mannose [21] and is encoded by the YlHXK1 (YALI0B22308g) gene. The primary structure of YlHxk1 has quaint characteristics absent in other known hex- okinases. In addition to conserved domains allowing for the action on hexoses, YlHxk1 contains an atypical 37-amino acid loop spanning between glucose- and ATP-binding domains [21], which indirectly influences enzyme activity [22]. The overexpression of YlHXK1 enhanced fructose utilization in the Y. lipolytica strains showing tedious growth on this monosaccharide, such as the wild strain W29, and more generally improved hexose utilization, increased carbon flux through glycolysis, changed the preferential use of glyc- erol to glucose when both substrates were present and reduced filamentation [20,22,23]. It also boosted lipid biosynthesis from fructose in an obese Y. lipolytica strain up to 55% [24]. The overexpression had, however, no effect on preferential uptake of glucose over fructose. The preference might in turn result from the kinetic properties of YlHxk1 as the enzyme has higher affinity for glucose than for fructose with a Km of 0.38 mM and 3.56 mM, respec- tively [21], even more pronounced than for the ScHxk2 hexokinase of S. cerevisiae (0.25 mM for glucose; 1.5 mM for fructose). In contrast, hexokinase from Schizosaccharomyces pombe (SpHxk1) is fructophilic with a Km of 8.4 mM for glucose and 1.5 mM for fructose [25]. In this study we investigated the role of hexose transporters and hexokinase in the preferential uptake of glucose over fructose in Y. lipolytica. We first examined the kinetic properties of Yht1, Yht3 and Yht4 in a heterologous host and then, through overexpression of hexose transporters in combination with native YlHxk1 or heterologous SpHxk1, we showed rearrangement of glucose and fructose utilization profiles. We further indicated that the increased carbon flux can be directed to different metabolic pathways by changing cultivation conditions. In addition, we propose that the introduced modifications can be harnessed for production of polyols by Y. lipolytica grown in hexose-containing media. 2. Results 2.1. Expression of Y. lipolytica Hexose Transporters in Heterologous Host Induce Diverse Monosaccharide Utilization Phenotypes The heterologous host S. cerevisiae EBY.VW4000 devoid of hexose transporters [26] is well-suited for comparing hexose uptake characteristics conferred by single transporters and has been previously used for this purpose [20,27,28]. The S. cerevisiae strains expressing YHT1, YHT3H222 (from Y. lipolytica H222, named hereafter YHT3) or YHT4 were cultivated Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 20 2. Results 2.1. Expression of Y. lipolytica Hexose Transporters in Heterologous Host Induce Diverse Monosaccharide Utilization Phenotypes The heterologous host S. cerevisiae EBY.VW4000 devoid of hexose transporters [26] is well-suited for comparing hexose uptake characteristics conferred by single transporters
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